Many growth factors and hormones such as nerve growth factor (NGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and insulin mediate their signals through interactions with cell surface tyrosine kinase receptors. The transduction of extracellular signals across the membrane, initiated by ligand binding, leads to the propagation of multiple signaling events which ultimately control target biochemical pathways within the cell.
The phosphatidylinositol 3-kinases (PI3Ks) represent a ubiquitous family of heterodimeric lipid kinases that are found in association with the cytoplasmic domain of hormone and growth factor receptors and oncogene products. PI3Ks act as downstream effectors of these receptors, are recruited upon receptor stimulation and mediate the activation of second messenger signaling pathways through the production of phosphorylated derivatives of inositol (Fry, Biochim. Biophys. Acta., 1994, 1226, 237-268).
PI3Ks have been implicated in many cellular activities including growth factor mediated cell transformation, mitogenesis, protein trafficking, cell survival and proliferation, DNA synthesis, apoptosis, neurite outgrowth and insulin-stimulated glucose transport (reviewed in Fry, Biochim. Biophys. Acta., 1994, 1226, 237-268).
The PI3 kinase enzyme heterodimers consist of a 110 kD (p110) catalytic subunit associated with an 85 kD (p85) regulatory subunit and it is through the SH2 domains of the p85 subunit that the enzyme associates with the membrane-bound receptors (Escobedo et al., Cell, 1991, 65, 75-82; Skolnik et al., Cell, 1991, 65, 83-90).
PI3K p85 (also known as GRB1 and PIK3R1) was initially isolated from bovine brain and shown to exist in two forms, a and .beta.. In these studies p85 isoforms were shown to bind to and act as substrates for tyrosine-phosphorylated receptor kinases and the polyoma virus middle T antigen complex (Otsu et al., Cell, 1991, 65, 91-104). Since then the p85 subunit has been further characterized and shown to interact with a diverse group of proteins including tyrosine kinase receptors such as the erythropoietin receptor, the PDGR-.beta. receptor and Tie2, an endothelium-specific receptor involved in vascular development and tumor angiogenesis (Escobedo et al., Cell, 1991, 65, 75-82; He et al., Blood, 1993, 82, 3530-3538; Kontos et al., Mol. Cell. Biol., 1998, 18, 4131-4140). It also interacts with focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase involved in integrin signaling and is thought to be a substrate and effector of FAK. The p85 subunit also interacts with profilin, an actin binding protein that facilitates actin polymerization (Bhargavi et al., Biochem. Mol. Biol. Int., 1998, 46, 241-248; Chen and Guan, Proc. Natl. Acad. Sci. U.S.A., 1994, 91, 10148-10152), and the p85/profilin complex inhibits actin polymerization.
Recently, a truncated form of the PI3K p85 subunit was solated (Jimenez et al., Embo J., 1998, 17, 743-753). This orm includes the first 571 amino acids of the wild type (encoded by nucleotides 43-1755 of Genbank Acc. No. M61906) linked to a region that is conserved in the eph tyrosine kinase receptor family. This truncation was shown to induce the constitutive activation of PI3 kinase and contribute to cellular transformation of mammalian fibroblasts.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of PI3 kinase and the major approach to studying PI3 kinase function has been the use of inhibitors.
Several chemically distinct inhibitors for PI3 kinases are reported in the literature. These include wortmannin, a fungal metabolite (Ui et al., Trends Biochem. Sci., 1995, 20, 303-307); demethoxyviridin, an antifungal agent (Woscholski et al., FEBS Lett., 1994, 342, 109-114) and quercetin and LY294002, two related chromones (Vlahos et al., J. Biol. Chem., 1994, 269, 5241-5248). However, these inhibitors primarily target the p110 subunit and are untested as therapeutic protocols. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting P13K p85 function.
Alternatively, antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of PI3K p85 expression.
The present invention provides compositions and methods for modulating PI3K p85 expression, including modulation of the truncated form of PI3K p85.